Process of chiral resolution of cyclic and acyclic acetates to enantiomerically pure (R)-alcohols

Abstract

The patent discloses herein a process for the chiral resolution of racemic cyclic and acyclic acetates to obtain (R)-alcohol. Further, it discloses the resolution of racemic cyclic and acyclic acetates to obtain enantiomerically pure (R)-()-alcohol as single enantiomer through fungal catalyzed deacylation in single fermentation, wherein fungal strains are F. proliferatum.

Claims

1. A process for chiral resolution of a racemic acetate to obtain an enantiomerically pure (R)-Alcohol as a single enantiomer comprising the steps of: i) incubating Fusarium proliferatum for from 24 to 48 hours at a temperature between 28 C. to 30 C. in a medium; ii) adding the racemic acetate to the medium after step i) and incubating further for from 6 hours to 3 days at temperature between 28 C. to 30 C. to obtain the enantiomerically pure (R)-Alcohol.

2. The process according to claim 1, wherein the Fusarium proliferatum comprises spores and mycelia.

3. The process according to claim 1, wherein the racemic acetate is selected from the group consisting of 2-Heptyl acetate, lavandulyl acetate and 2-Hexyl acetate.

4. The process according to claim 1, wherein the racemic acetate is a cyclic racemic acetate or an acyclic racemic acetate.

5. The process according to claim 1, wherein a yield of the enantiomerically pure (R)-Alcohol as a single enantiomer is 95-99.9%.

6. The process according to claim 1, wherein the enantiomerically pure (R)-Alcohol is selected from the group consisting of R-lavandulol, R-2-Hexnol, R-2-Heptanol, R-1-Phenyl ethanol and R-1-Phenyl propanol.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a GC chromatogram showing conversion of ()-2-Hexyl acetate to R-()-2-Hexanol by Resting cell experiment over a period of 6 hrs. by F. proliferatum.

(2) FIG. 2 is a GC chromatogram showing conversion of ()-2-Heptyl acetate to R-()-2-Heptanol by Resting cell experiment over a period of 6 hrs. by F. proliferatum.

(3) FIG. 3 is a GC chromatogram showing Resting cell experiment with ()-Lavandulyl acetate.

(4) FIG. 4 is a GC chromatogram showing Resting cell experiment with S-()-Lavandulyl acetate.

(5) FIG. 5 is a GC chromatogram showing Resting cell experiment with S-(+)-2-Hexyl acetate.

(6) FIG. 6 is a GC chromatogram showing Resting cell experiment with S-(+)-2-Heptyl acetate.

(7) FIG. 7 is a GC chromatogram showing Resting cell experiment with ()-2-Hexanol.

(8) FIG. 8 is a Mass Fragmentation of (R)-()-2-Hexanol produced by F. proliferatum after conversion.

(9) FIG. 9 is a Mass Fragmentation of 2-Hexanone produced by F. proliferatum after conversion.

(10) FIG. 10 is a Mass Fragmentation of (R)-()-2-Heptanol produced by F. proliferatum after conversion.

(11) FIG. 11 is a Mass Fragmentation of 2-Heptanone produced by F. proliferatum after conversion.

(12) FIG. 12 is a GC chromatogram showing consumption of 2-Heptanone by F. proliferatum after 6, 12, 18 and 24 hrs.

(13) FIG. 13 is a GC-MS chromatogram showing consumption of single isomer (S) of Lavandulol. GC-MS chromatograms showing consumption of (S)-isomer of Lavandulol by F. proliferatum over a period of 1, 3 and 5 days.

(14) FIG. 14 is a Mass spectra of (R)-Lavandulol [].sub.D=10.12 (c=3 Chloroform), Mass spectra along with library search of (R)-Lavandulol

(15) FIG. 15 is a GC chromatogram showing Resting cell experiment with ()-1-Phenylethyl acetate.

(16) FIG. 16 is a GC chromatogram showing Resting cell experiment with ()-1-Phenylpropyl acetate.

DETAILED DESCRIPTION OF THE INVENTION

(17) In view of above, the present invention provides an easier and cheaper process of producing enantiomerically pure R-Alcohols from racemic cyclic and acyclic acetates by whole cell method.

(18) As used herein the term chiral resolution means not only separation but also going from racemic to single enantiomer.

(19) According to the invention, whole cell microorganisms mean bacteria or fungal organisms or other microorganisms known to produce lipases t Alcohol dehydrogenases.

(20) In the present invention, fungal strains such as F. proliferatum is used for resolving racemic cyclic and acyclic acetates into (R)-Alcohols. Thus, this process can be used for the production of (R)-Alcohol from the racemic cyclic and acyclic acetate.

(21) In an embodiment, the invention provides resolution of racemic cyclic and acyclic acetate to obtain enantiomerically pure (R)-Alcohol as single enantiomer through fungal catalyzed deacylation in single fermentation, wherein fungal strains selected is F. proliferatum.

(22) The chiral resolution of racemic 2-Hexyl acetate to obtain enantiomerically pure (R)-2-Hexanol as a single enantiomer is represented as below Scheme 1:

(23) ##STR00004##

(24) The chiral resolution of racemic 2-Heptyl acetate to obtain enantiomerically pure (R)-2-Heptanol as a single enantiomer is represented as below in Scheme 2:

(25) ##STR00005##

(26) The chiral resolution of racemic lavandulyl acetate to obtain enantiomerically pure (R)-lavandulol as a single enantiomer is represented as depicted below in Scheme 3:

(27) ##STR00006##

(28) The chiral resolution of racemic 1-Phenylethyl acetate to obtain enantiomerically pure (R)-1-Phenylethanol as a single enantiomer is represented as depicted below in Scheme 4:

(29) ##STR00007##

(30) The chiral resolution of racemic 1-Phenylpropyl acetate to obtain enantiomerically pure (R)-1-Phenylpropanol as a single enantiomer is represented as depicted below in Scheme 5:

(31) ##STR00008##

(32) In the present invention, the other isomer (S)-Alcohol has been converted to corresponding ketone and then it is further consumed by the fungus.

(33) The preferential use of whole cells over enzymes as biocatalysts in the production of useful organic compounds mostly results from the costs of enzyme isolation, purification and stabilization in the latter method.

(34) This method is simple and very useful for the production of (R)-Alcohols in large scale. Also it is cost effective as the process involves whole cell microorganisms. Advantage is no isolation and no purification needed.

(35) Resting cell experiments showed that well grown fungi in modified Czapek Dox medium were able to resolve of racemic 2-Hexyl acetate (0.005 g), 2-Heptyl acetate (0.005 g), Lavandulyl acetate (0.005 g), 1-Phenylethyl acetate (0.005 g) and 1-Phenylpropyl acetate (0.005 g) in to (R)-2-Hexanol, (R)-2-Heptanol, (R)-Lavandulol, (R)-1-Phenylethanol and (R)-1-Phenylpropanol respectively in quantitative yield with respect to the ratio of racemic mixture

(36) This microorganism requires 6 hrs of incubation in resting cell experiments with 2-Hexyl acetate and 2-Heptyl acetate while racemic lavandulyl acetate requires 3 days for the complete transformation of by whole cell method and 24 hrs. by resting cell method. The remaining (S)-isomer of the alcohol has been converted to corresponding ketone by the organism.

(37) GC-MS studies using chiral column showed the presence of only (R)-lavandulol and not any other metabolites.

EXAMPLES

(38) Following examples are given by way of illustration therefore should not be construed to limit the scope of the invention.

Example 1

(39) Enantiopure Preparation of R-()-Lavandulol from ()-Lavandulyl Acetate Using F. Proliferatum by Whole Cell Method

(40) Into a 250 mL conical flask, 50 mL of modified Czapex Dox (C.Z) media pH-5.8 was taken and spores (10.sup.9 per mL) of F. proliferatum (Deposition details: Fusarium proliferatum (NCIM cat No. 1105), OCT-F-25, Accession no: MCC0011) in water were added to it. Flasks were incubated for 48 hrs at temp 30 C. for their complete growth. In 3 flasks each containing fully grown fungus, substrate ()-Lavandulyl acetate was added at concentration of 30 mg/50 mL) into it. Microorganism and substrate controls were also kept along with it( ). These flasks were incubated in incubator shaker at 30 C. and 200 rpm for 3 days and they were extracted at the interval of 1, 2, 3 days and analyzed by GC on Chiral column. At the end of 3 days, R-Lavandulol with more than 99% enantiopurity is formed. The confirmation study was done by co-injection of R-Lavandulol with extracted sample.

Example 2

(41) Enantiopure Preparation of R-()-Lavandulol from ()-Lavandulyl Acetate Using F. Proliferatum by Resting Cell Experiment

(42) Into a 250 mL conical flask, 50 mL of Potassium phosphate buffer with pH-7.2 in addition of 0.2% glucose was taken. 4 flasks each containing 2 gm of fungal mycelia for 50 mL of buffer was taken and the compound ()-Lavandulyl acetate (5 mg) was added into it. Microorganism and substrate controls were also kept along with it.

(43) These flasks were incubated in incubator shaker at 30 C. and 200 rpm. 4 Flasks along with controls were extracted at the interval of 6, 12, 18 and 24 hrs. and analyzed by GC on Chiral column. R-Lavandulol with more than 99% enantiopurity is formed at the end of 24 hrs. The confirmation study was done by co-injection of R-Lavandulol with extracted sample

Example 3

(44) Enantiopure Preparation of R-()-2-Hexanol from ()-2-Hexyl Acetate Using F. Proliferatum by Resting Cell Experiment

(45) Into a 250 mL conical flask, 50 mL of Potassium phosphate buffer with pH-7.2 in addition of 0.2% glucose was taken. 4 flasks each containing 2 gm of fungal mycelia for 50 mL of buffer was taken and the compound ()-2-Hexyl acetate (5 mg) was added into it. Microorganism and substrate controls were also kept along with it. These flasks were incubated in incubator shaker at 30 C. and 200 rpm. 4 Flasks along with controls were extracted at the interval of 1, 2, 3, 4, 5 and 6 hrs. and analyzed by GC on Chiral column. R-2-Hexanol with more than 99% enantiopurity is formed at the end of 6 hrs. The confirmation study was done by co-injection of R-2-Hexanol with extracted sample

Example 4

(46) Enantiopure Preparation of R-()-2-Heptanol from ()-2-Heptyl Acetate Using F. proliferatum by Resting Cell Experiment

(47) Into a 250 mL conical flask, 50 mL of Potassium phosphate buffer with pH-7.2 in addition of 0.2% glucose was taken. 4 flasks each containing 2 gm of fungal mycelia for 50 mL of buffer was taken and the compound ()-2-Heptyl acetate (5 mg) was added into it. Microorganism and substrate controls were also kept along with it.

(48) These flasks were incubated in incubator shaker at 30 C. and 200 rpm. 4 Flasks along with controls were extracted at the interval of 1, 2, 3, 4, 5 and 6 hrs. and analyzed by GC on Chiral column. R-2-Heptanol with more than 99% enantiopurity is formed at the end of 6 hrs. The confirmation study was done by co-injection of R-2-Heptanol with extracted sample.

Example 5

(49) Enantiopure Preparation of R-(+)-1-Phenyl Ethanol from ()-1-Phenyl Ethyl Acetate Using F. Proliferatum by Resting Cell Experiment

(50) Into a 250 mL conical flask, 50 mL of Potassium phosphate buffer with pH-7.2 in addition of 0.2% glucose was taken. 4 flasks each containing 2 gm of fungal mycelia for 50 mL of buffer was taken and the compound ()-1-Phenylethyl acetate (5 mg) was added into it. Microorganism and substrate controls were also kept along with it. These flasks were incubated in incubator shaker at 30 C. and 200 rpm. 4 Flasks along with controls were extracted at the interval of 2, 4, 6, 8, 10 and 12 hrs. and analyzed by GC on Chiral column. R-(+)-1-Phenyl ethanol with more than 99% enantiopurity is formed at the end of 12 hrs. The confirmation study was done by co-injection of R-(+)-1-Phenyl ethanol with extracted sample.

Example 6

(51) Enantiopure Preparation of R-(+)-1-Phenyl Propanol from ()-1-Phenyl Propyl Acetate Using F. Proliferatum by Resting Cell Experiment

(52) Into a 250 mL conical flask, 50 mL of Potassium phosphate buffer with pH-7.2 in addition of 0.2% glucose was taken. 4 flasks each containing 2 gm of fungal mycelia for 50 mL of buffer was taken and the compound ()-1-Phenylpropyl acetate (5 mg) was added into it. Microorganism and substrate controls were also kept along with it. These flasks were incubated in incubator shaker at 30 C. and 200 rpm. 4 Flasks along with controls were extracted at the interval of 6.12, 18 and 24 hrs. and analyzed by GC on Chiral column. R-(+)-1-Phenyl propanol with more than 99% enantiopurity is formed at the end of 24 hrs. The confirmation study was done by co-injection of R-(+)-1-Phenyl propanol with extracted sample.

ADVANTAGES OF INVENTION

(53) a. This method is simple and very useful for the production of (R)-Alcohols in large scale. b. Also it is cost effective as the process involves whole cell microorganisms. c. No isolation and no purification needed. d. The process can be used for Lavandulol which is an important constituent of essential oils and has been identified as a sex pheromone. e. The process can be used for R-2-Hexanol/R-2-Heptanol which are important fragrance compounds and pharmaceutical intermediates used in various drug preparations. f. The process can be used for R-1-Phenyl ethanol/R-1-Phenyl propanol which are very much important chiral drug intermediates and flavor compounds used in pharmaceutical and fragrance industry.